Rat Mammary carcinoma susceptibility 3 (Mcs3) pleiotropy, socioenvironmental interaction, and comparative genomics with orthologous human 15q25.1-25.2

Abstract Genome-wide association studies of breast cancer susceptibility have revealed risk-associated genetic variants and nominated candidate genes; however, the identification of causal variants and genes is often undetermined by genome-wide association studies. Comparative genomics, utilizing Rattus norvegicus strains differing in susceptibility to mammary tumor development, is a complimentary approach to identify breast cancer susceptibility genes. Mammary carcinoma susceptibility 3 (Mcs3) is a Copenhagen (COP/NHsd) allele that confers resistance to mammary carcinomas when introgressed into a mammary carcinoma susceptible Wistar Furth (WF/NHsd) genome. Here, Mcs3 was positionally mapped to a 7.2-Mb region of RNO1 spanning rs8149408 to rs107402736 (chr1:143700228–150929594, build 6.0/rn6) using WF.COP congenic strains and 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis. Male and female WF.COP-Mcs3 rats had significantly lower body mass compared to the Wistar Furth strain. The effect on female body mass was observed only when females were raised in the absence of males indicating a socioenvironmental interaction. Furthermore, female WF.COP-Mcs3 rats, raised in the absence of males, did not develop enhanced lobuloalveolar morphologies compared to those observed in the Wistar Furth strain. Human 15q25.1-25.2 was determined to be orthologous to rat Mcs3 (chr15:80005820–82285404 and chr15:83134545–84130720, build GRCh38/hg38). A public database search of 15q25.1-25.2 revealed genome-wide significant and nominally significant associations for body mass traits and breast cancer risk. These results support the existence of a breast cancer risk-associated allele at human 15q25.1-25.2 and warrant ultrafine mapping of rat Mcs3 and human 15q25.1-25.2 to discover novel causal genes and variants.


Introduction
Breast cancer is the most frequently diagnosed cancer among women and a leading cause of premature mortality (DeSantis et al. 2014;Oeffinger et al. 2015). Susceptibility to this complex disease is modulated by genetic, epigenetic, and environmental factors. The genetic component of breast cancer susceptibility is controlled by an array of high, moderate, and low penetrance risk-associated mutations and variants (Pharoah et al. 2002). Breast cancer susceptibility is a polygenic model with many genes contributing. High to moderate penetrant mutations in breast cancer predisposition genes, such as BRCA1 and BRCA2, are rare in large populations and account for only 5-10% of total breast cancer cases (Carroll et al. 2008;Economopoulou et al. 2015). A majority of genetic risk is accounted for by additive effects of low-penetrant variants in noncoding DNA regulatory elements (Breast Cancer Association Consortium 2006;Mavaddat et al. 2010). Causal genes at most susceptibility loci remain unknown (Breast Cancer Association Consortium 2006;Smith et al. 2006;Ghoussaini and Pharoah 2009;Mavaddat et al. 2010;Michailidou et al. 2017). In addition to difficulty in determining causal genes, many nominally associated variants are identified in genome-wide association studies (GWAS) that do not meet stringent P-values required for genome-wide significance. These associations mark potential true positive associations. One way to evaluate these nominally associated alleles and identify causal genes is with comparative genomics approaches that incorporate experimental organisms that models breast cancer susceptibility (Gould 2009;Colletti et al. 2014;Sanders and Samuelson 2014).
The laboratory rat (Rattus norvegicus) presents one of the best in vivo human breast cancer models, as induced rat mammary and female breast carcinomas have similar histopathological stages and features, including epithelial ductal cell origin, progression, and hormone responsiveness and nonresponsiveness (Russo et al. 1990;Nandi et al. 1995;Russo and Russo 1996;Shepel et al. 1998;Samuelson et al. 2007;Gould 2009;Sharma et al. 2011). Inbred rat strains differ in their susceptibility to 7,12-dimethylbenz(a)anthracene (DMBA)-, N-methyl-N-nitrosourea-, and estrogen-induced mammary carcinogenesis. Many rat strains have been used to discover quantitative trait loci (QTLs) that control mammary cancer susceptibility. These loci are named Mammary carcinoma susceptibility (Mcs) and Estrogen-induced mammary cancer (Emca) QTLs (Hsu et al. 1994;Shepel et al. 1998;Lan et al. 2001;Gould et al. 2004;Quan et al. 2006;Schaffer et al. 2006;Ren et al. 2013). Genetic linkage analyses of crosses between DMBA-induced mammary carcinoma susceptible Wistar Furth (WF) and resistant Copenhagen (COP) strains resulted in 4 predicted Mcs QTLs termed Mcs1, Mcs2, Mcs3, and Mcs4 (Hsu et al. 1994;Shepel et al. 1998). The Mcs1, Mcs2, and Mcs3 QTLs were physically confirmed using congenic rat strains with the COP resistance-associated allele of interest introgressed into a susceptible WF genome (Haag et al. 2003;Sanders et al. 2011;Le et al. 2017). Rat Mcs3 has been physically confirmed as an independently acting QTL and delimited to a 29.4-Mb genomic region of rat chromosome one (RNO1) (Le et al. 2017). In this article, we report WF.COP congenic strain studies that further delimit the Mcs3 resistance allele to a 7.2-Mb region of RNO1. We also report that Mcs3 females have reduced body mass and different mammary gland morphology compared to WF females. Interestingly, these latter phenotypes only manifest when females are housed in the absence of males. In our comparative genomics analysis of Mcs3 and orthologous human 15q25.1-25.2, we found previously reported breast cancer and body mass-associated variants.

Methods
Congenic rat strains were established and maintained in an Association for the Assessment and Accreditation of Laboratory Animal Care-approved facility on a 12-h light/dark cycle and provided LabDiet 5001 Rodent Diet (PMI Nutrition International) and water ad libitum. The University of Louisville Animal Care and Use Committee approved all animal protocols used in this study. Rat Mcs3 WF.COP congenic strains were developed with a WF/ NHsd genome and COP/NHsd alleles introgressed at selected loci spanning the previously published Mcs3 QTL (Le et al. 2017). Rat WF.COP-Mcs3 resistance-associated strain D had a COP allele spanning RN01 from single-nucleotide variant (SNV) rs8149408 to SNV rs105131702 (Le et al. 2017). Heterozygous (COP/WF) WF.COP-Mcs3 D males at congenic generation N16 were backcrossed to inbred WF/NHsd females (Envigo). Informative N17 recombinants were backcrossed to WF/NHsd animals for expansion, and subsequent N18 offspring were inbred using brothersister matings to establish unique WF.COP-RNO1 congenic strains for this study. Sequence information and genomic locations of genetic markers defining COP alleles in these strains are available at the UCSC Genome Browser (www.genome.ucsc.edu), the Rat Genome Database (http://rgd.mcw.edu/), and Supplementary Tables 1 and 2. A WF.COP strain that did not inherit COP alleles at the N18 generation was maintained in house to serve as a WF/ NHsd mammary carcinoma susceptible strain.
Genotyping was completed using standard PCR-based genotyping methods previously described (Samuelson et al. 2003). Briefly, microsatellite marker primers were used to PCR amplify genomic DNA at the congenic allelic segment ends and intervals within. Fast-PCRs underwent denaturation of 95 C for 10 s, followed by 40 cycles of 94 C for 0 s and 63 C for 8 s, and an extension at 72 C for 30 s on an Applied Biosystems Veriti Fast Thermal Cycler. Amplified DNA was resolved on 3% high-resolution agarose gels, stained with ethidium bromide or SybrGold, scanned with a digital imager, and visualized with ImageQuant (Amersham Biosciences). Segments containing 4 WF/COP informative SNVs were PCR-amplified and sequenced using dideoxy sequencing by the University of Louisville, Center for Genetics and Molecular Medicine DNA sequencing core with an ABI PRISM 3130XL Sequence Detection System (Life Technologies).
To measure body mass, congenic WF.COP-Mcs3 strain J (Mcs3 J ) and WF/NHsd male and female rats in regular housing with both males and females present were weighed at 4, 8, and 12 weeks of age (n ¼ 31 WF males, n ¼ 34 WF females, n ¼ 30 Mcs3 J males, and n ¼ 29 Mcs3 J females) using a standard digital scale. In a follow-up experiment, WF.COP-Mcs3 J (n ¼ 24) and WF/NHsd (n ¼ 15) females were housed without males present in the room after weaning and weighed at 8 and 12 weeks of age. The congenic generations of these rats were N18-F3, -F4, and -F5.
Mammary gland lobuloalveolar morphology was quantified in FFPE, H&E-stained mammary gland cross-sections from 12week-old WF/NHsd (N18F10) and Mcs3 J (N18F9) females that had been housed in regular housing with either males present or absent after weaning (n ¼ 3 rats per group). Tissue morphology of interest was outlined, and % area quantified using open-source ImageJ Fiji software. Averages of 3 representative fields of view per rat were used for analysis.
Comparative genomics involved interrogating the rat Mcs3 and human orthologous sequences. Human orthologous syntenic regions that mapped to the delimited rat Mcs3 were identified using the convert function of the UCSC Genome Browser. The R. norvegicus reference genome build version RGSC 6.0/rn6 and Homo sapiens version GRCh38/hg38 were used. Rat Mcs3 and human orthologous syntenic region annotated genes and noncoding DNA were curated using the table browser function of the UCSC Genome Browser. The NHGRI-EBI Catalog of human genomewide association studies was used to identify breast cancer disease and risk correlated traits that had P-values of 1 Â 10 À7 or less for association. NCBI-PubMed was searched to identify rat Mcs3 and human orthologous genes with published associations to breast cancer.
Statistical analysis of mammary carcinoma multiplicity data was performed using nonparametric Kruskal-Wallis and Dunn's post hoc tests. Body mass data collected at the time of mammary tumor multiplicity counts were analyzed by ANOVA and Dunnett's post hoc test. Body mass data collected at different ages were analyzed by 2-way ANOVA with strain and age as independent variables, followed by Tukey's post hoc test. Mammary gland lobuloalveolar cross-sectional area percentage quantifications were arcsine transformed and analyzed using a 2-tailed unpaired t-test. P-values 0.05 were considered statistically significant. All statistical analyses were done using GraphPad Prism version 9.2.0 for Windows, GraphPad Software, La Jolla, CA, USA. All data are presented within the article and supplementary files.

Mapping and pleiotropy of Mcs3
Rat Mcs3 was physically mapped using 4 WF.COP congenic strains that contained different COP donor segments of RNO1 (Fig. 1). Together these segments covered a 29.4-Mb region of RNO1 that previously defined Mcs3 (Shepel et al. 1998;Le et al. 2017). Genomic locations and genetic markers of COP alleles contained in each WF.COP congenic strain along with respective mammary tumor multiplicity phenotypes are in Table 1. Mammary carcinoma susceptibility phenotype statistics of these WF.COP and WF/NHsd strains are displayed graphically in Fig. 2 and Supplementary Fig. 1. The congenic recipient strain (WF/NHsd) is known to have a high susceptibility phenotype. These females (n ¼ 28) developed an average of 7.9 6 2.9 (mean 6 SD) mammary tumors per rat in this study. This phenotype was similar to previously published phenotypes for this strain (Veillet et al. 2011;denDekker et al. 2012;Le et al. 2017). WF.COP congenic strains H, I, and K developed 6.7 6 3.4 (n ¼ 20), 5.9 6 3.8 (n ¼ 26), and 7.0 6 3.5 (n ¼ 25) mammary tumors per rat, respectively. None of these strains were statistically different in susceptibility from the WF/ NHsd strain (P-values ¼ 0.766, 0.0932, and >0.999, respectively). WF.COP strain J females (n ¼ 30) developed 3.4 6 2.7 mammary tumors per rat, which was significantly different than WF/NHsd females (P < 0.0001). Collectively, this congenic strain panel delimited rat Mcs3 to a 7.2-Mb region of RNO1 spanning from genetic markers rs8149408 to rs107402736 (RNO1:143700228-150929594, rat reference genome build RGSC 6.0/rn6).
Histopathological analysis of mammary tumors revealed no discernable differences between WF.COP congenic and WF strains. Mammary tumors were invasive ductal carcinomas, with a majority being invasive papillary and invasive cribriform carcinoma subtypes (Fig. 3).
Pleiotropy of the Mcs3 allele contained in WF.COP strain J (Mcs3 J ) was observed in this study. Rat Mcs3 J females, exposed to DMBA, had a body mass of 179 6 17 g (mean 6 SD) at 23 weeks of age, which was significantly lower than age-matched and DMBA-exposed WF/NHsd females that had a body mass phenotype of 201 6 21 g (P < 0.0001) (Fig. 4a). Mammary cancer-susceptible WF.COP congenic strains K, H, and I had body mass phenotypes of 210 6 13, 210 6 11, and 202 6 13 g, respectively. These phenotypes were not different from the WF/NHsd phenotype (P-values ¼ 0.4788, 0.9560, and >0.999, respectively).
We measured body mass of male and female Mcs3 J and WF rats at 4, 8, and 12 weeks of age to determine the effect of Mcs3 at different stages of male and female development ( Fig. 4b) (Supplementary Table 3). Respectively, these ages coincided with prepuberty, puberty, and adult stages of development. For these experiments, male and female rats were housed in separate cages, but within the same environment (standard housing room) and not exposed to DMBA. There was no significant difference between strains for either biological sex at 4 weeks of age. Interestingly, Mcs3 J females housed in rooms containing males and not exposed to DMBA had body mass phenotypes at 4, 8, and 12 weeks of age that were not different from WF females. However, at 8 weeks of age, males homozygous for Mcs3 J alleles had a body mass of 149 6 21 g (n ¼ 30), which was significantly less than the WF male body mass of 186 6 15 g (n ¼ 31) (P < 0.0001). A significant difference between Mcs3 J and WF male body mass was also observed at 12 weeks of age (244 6 37 and 267 6 29 g, respectively, P < 0.0001).
Females in rat mammary carcinogenesis studies were housed in environments that did not contain males. We questioned whether the effect of Mcs3 on body mass seen at 23 weeks of age in DMBA-exposed females was due to the absence of males (Fig. 4a). To test this, we raised females in the absence males postweaning, but did not administer DMBA (Fig. 4c). In the absence of males, homozygous Mcs3 J females (n ¼ 24) had significantly lower body mass compared to WF/NHsd females (n ¼ 15) at 8 weeks of age (114 6 9 and 134 6 5 g, respectively, P < 0.0001) and at 12 weeks of age (152 6 13 and 178 6 7 g, respectively, P < 0.0001) (Fig. 4c). Body mass statistics are contained in Supplementary Table 3.
Histological analysis was completed to determine if the Mcs3 J allele influenced mammary gland development or morphology. Abdominal-inguinal mammary glands at 4, 8, and 12 weeks of age from females raised in the presence or absence of males were analyzed. Females from both Mcs3 J and WF/NHsd strains in both housing conditions had similar terminal end bud (TEB) and ductal structure morphologies at 4 and 8 weeks of age with both having more pronounced stroma at 8 weeks (Figs. 5a/b and 6a/b). At 12 weeks of age, WF/NHsd and Mcs3 J females raised in the presence of males exhibited loss of TEB structures, largely ductal histology, and little lobuloalveolar epithelium as typical of the adult virgin mammary gland (Fig. 5c). There was no quantitative difference in lobuloalveolar morphological area between strains (Fig. 5d). Adult Mcs3 J and WF/NHsd females raised in the absences of males had dramatically different mammary gland histology at 12 weeks of age (Fig. 6c). The mammary cancer-susceptible WF strain had a more enhanced lobuloalveolar morphology, which was not observed in Mcs3 J females. Quantification of H&Estained mammary gland cross-sections revealed that the WF strain had 6-times more lobuloalveolar morphological area compared to Mcs3 J females at 12 weeks of age (P < 0.0001, Fig. 6d).

Comparative genomics of rat Mcs3
Rat Mcs3 (7.2 Mb, rs8149408 to rs107402736) was found to contain sequence orthologous to segments of human chromosomes 11 and 15 (Table 2). Rat Mcs3 also contained genic sequence that mapped to gene orthologs on human chromosome 10 (Table 3).
Rat and human reference genomes were searched to identify annotated genes in rat Mcs3 and human orthologous regions. There were 23 genes in common between rats and humans, 12 human-only, and 20 rat-only genes. A majority of the rat-only genes encode olfactory receptors not conserved in the human genome. Eleven of the 23 genes in common had published   Fig. 7. Human genome segments orthologous to rat Mcs3 were queried for genetic associations to breast disease and cancer risk correlated traits using the NHGRI-EBI catalog of human genome-wide association studies. Human 15q25.2 variant rs6495623 (hg38 chr15:81848308) was nominally associated to breast cancer risk with a p-value for association of 0.000871 (Hunter et al. 2007). Two of the human syntenic regions at 15q25.1-25.2 (chr15:80005820-82285404 and chr15:83134545-84130720) possessed 69 relevant body mass-associated variants (P-value for association <10 À7 ) that were reported in genetic association studies of body mass index, visceral adipose tissue measurement, waist-hip ratio, BMI-adjusted hip circumference, BMI-adjusted waist circumference, lean body mass, body fat distribution, and body fat percentage (Fig. 8 and Supplementary Table 4).

Discussion
Rat Mcs3 was a previously mapped and physically confirmed mammary carcinoma resistance allele that, in this study, was further delimited from a 29.4-to 7.2-Mb segment of RNO1 with orthology to 3 regions of the human genome. One of these segments, human 15q25.2, contains a nominally associated breast cancer risk variant (rs6495623, Hunter et al. 2007). A variant located in 15q25.1, rs149479659, is associated with ERþ breast cancer mortality (Escala-Garcia et al. 2019). Eight out of 11 genes located within rat Mcs3 and human 15q25.1-25.2 orthologous sequence were found to have previously published functional associations to breast cancer (Table 3). Interestingly, human 15q25.1 variation has been associated with lung cancer susceptibility (Amos et al. 2008;Hung et al. 2008;Gu et al. 2012). The possibility of a genetic correlation between breast and lung cancer (r g ¼ 0.   The bottom row contains representative papillary patterns, defined by fibrovascular cores that form a network throughout the tumor (arrow C), with neoplastic epithelial cells growing to expand the lesion via papillary projections (arrow D). Images were taken at 40Â magnification on an Aperio ImageScope CS2. our Mcs3 rat model could be used to investigate genetic mechanisms of breast and possibly lung cancer.
Rat Mcs3 did not influence mammary tumor histopathology, as both Mcs3 carcinoma resistance-associated and susceptible females developed papillary and cribriform mammary carcinomas. A majority of DMBA-induced rat mammary carcinomas are histologically defined as invasive papillary carcinomas and invasive cribriform carcinomas, comprising 32% and 27% of tumor histology, respectively (Russo 2015). A papillary histology is defined by branching fibrovascular cores throughout the lesion that contain variable degrees of stromal fibrosis and also support growth of papillary projections (Pal et al. 2010;Wei 2016;Rakha and Ellis 2018). A cribriform histology is defined by invasion of desmoplastic stroma by neoplastic epithelial cells that form nestlike tumor structures with a sieve-like appearance (Kadota et al. 2014;Russo 2015;Branca et al. 2017). Others have shown that DMBA-induced mammary tumors in pubescent, nulliparous rats, are adenocarcinomas of mammary ductal origin that develop primarily at TEBs and terminal ducts (Russo 2015). These intraductal and invasive ductal carcinomas are similar in histopathology to carcinomas observed in a majority of human breast malignancies (Russo 2015).
Mammary cancer-susceptible WF females had significantly higher lobuloalveolar morphology at 12 weeks compared to agematched resistant WF.COP-Mcs3 J females, but only when raised in the absences of males. This effect of the Mcs3 COP allele introgressed into a WF genome is supported by similar findings from a study of 17b-estradiol exposure that reported the inbred COP strain had reduced lobuloalveolar mammary epithelia compared to the mammary cancer-susceptible ACI strain (Harvell et al. 2000). Whether the complete inbred COP decreased lobuloalveolar development phenotype seen by Shull and colleagues is controlled by the Mcs3 locus remains to be determined. Our study mimicked effects of a continuous exposure to exogenous estradiol by housing females in socioenvironmental conditions without males present, which would be expected to cause hormone disruption. Although a causal relationship cannot be concluded from our study, it does suggest evidence to support the idea that a cancer-susceptible mammary gland may be innately more responsive to endogenous environmental components, such as hormonal perturbations, than a resistant mammary gland.
In addition to Mcs3 associated mammary cancer resistance and lobuloalveolar development, Mcs3 influenced body mass. Mammary cancer-resistant Mcs3 females had lower body mass than susceptible WF females. Body mass and genetically correlated traits, such as obesity, are associated with breast cancer risk (Kwan et al. 2012;Chan et al. 2014;Greenlee et al. 2017). Specifically, obesity is associated with larger tumor size, positive lymph node status, shorter time to disease recurrence, and mortality (Loi et al. 2005;Rosenberg et al. 2009;Chan et al. 2014;Copson et al. 2015). A positive correlation between obesity and breast cancer risk in postmenopausal women has been reported (Li et al. 2006;Neuhouser et al. 2015;Sebastiani et al. 2016;Picon-Ruiz et al. 2017). This relationship is hypothesized to be due to adipose tissue being a main site of estrogen synthesis in postmenopausal women (Brouckaert et al. 2018) and higher concentrations of estrogen that would be caused by excess fat reserves (Ooi et al. 2019). These greater estrogen levels are thought to drive estrogen-dependent tumors, which is supported by the fact that most breast cancer cases in postmenopausal obese women are ER positive (Enger et al. 2000;Rosenberg et al. 2006;Suzuki et al. 2006;Ahn et al. 2007;Canchola et al. 2012). Inversely, there is a negative relationship between obesity and breast cancer risk in premenopausal women (Michels et al. 2006;Berstad et al. 2010;Ooi et al. 2019). It has been speculated this might be due to less estrogen in circulation during menstrual cycling (Picon-Ruiz et al. 2017). It has been further suggested that this effect is reversed or lost upon adjusting for breast density (Boyd et al. 2006;Harris, Tamimi, et al. 2011;Engmann et al. 2019). Extra adipose tissue results in an inflammatory transcriptome and leads to the production of inflammatory cytokines, which creates a microenvironment conducive to cancer initiation, invasion, and metastasis (  females had lower body mass than susceptible WF/NHsd strain females at 23 weeks of age, which was when mammary carcinoma susceptibility phenotypes were measured (tumor multiplicity). A 1-way ANOVA was performed (P < 0.0001), followed by Dunnetts's post hoc tests comparing each strain to the control WF/NHsd strain. Strain J was significantly different from WF/NHsd (P < 0.0001). b) The effect of Mcs3 on female body mass was lost when females were raised in social environment containing males. Male and female rats from WF/NHsd and WF.COP-Mcs3 J strains were weighed at weaning age (4 weeks), puberty (8 weeks), and breeding age (12 weeks). WF.COP-Mcs3 J males had significantly reduced body mass compared to WF/NHsd males at 8 and 12 weeks of age (2-way ANOVA followed by Tukey's multiple comparisons post hoc test, P < 0.0001), but no differences in body mass were found between females at 4, 8, and 12 weeks of age. c) The effect of Mcs3 on body mass was evident at 8 and 12 weeks of age when, at weaning, females were placed in a social environment that did not contain males (2-way ANOVA, P < 0.0001).
as that observed in Mcs3 females, could be protective toward disease development. This would positively correlate with what has been found in human epidemiological studies of body mass and female breast cancer risk. Future studies of body mass differences between mammary cancer-susceptible and -resistant Mcs3 females could reveal body composition differences and corresponding physiological responses that contribute to mammary carcinoma resistance.
Many of the human GWAS-identified variants associated with body mass that are located within 15q25.1-25.2 reside in the SH3 Domain Containing GRB2 Like 3, Endophilin A3 (SH3GL3), and ADAMTS-like protein 3 (ADAMTSL3) genic region. This specific human gene region has not been shown to contain breast cancer risk-associated variants; however, the human body massassociated variants in this region are relevant because mammary cancer-resistant Mcs3 females had lower body mass than cancersusceptible females. This suggests that if Mcs3 controls both body mass and mammary cancer susceptibility, then these orthologous human regions may be doing the same.
The reduced body mass and mammary gland lobuloalveolar development phenotypes of Mcs3 females compared to cancersusceptible females was dependent on social environment, as Images were captured at 40Â magnification on an Aperio ImageScope CS2. a, b) Histology at 4 and 8 weeks is mixed population of TEBs and ductal structures embedded throughout an adipocyte matrix in both strains. c) Histology at 12 weeks is majority of mature ducts, with loss of TEBs, and minimal lobuloalveolar structures, which is representative of adult rat virgin mammary glands. d) Quantitative analysis of % lobuloalveolar morphology in mammary gland cross-sections at 12 weeks of age revealed no difference in lobuloalveolar area between WF/NHsd and WF.COP-Mcs3 J mammary glands [2-tailed unpaired t-test P > 0.05 (ns)].
Mcs3 females had a lower body mass and lobuloalveolar phenotypes only when housed in an environment without males present. The only social environmental difference was the presence or absence of male pheromones, as males were never housed within the same cage with experimental females after weaning. Male pheromones are known to induce physiological and hormonal changes in the female endocrine and reproductive system (Rekwot et al. 2001). For example, male urinary pheromones have been demonstrated to increase plasma-luteinizing hormone and progesterone levels in females (Tsai et al. 1994;Tomioka et al. 2005). The endocrine system has a role in body weight regulation. Disruptions of this system could theoretically influence overall body mass. Furthermore, regulatory action of ERa has a known downstream effect on body mass (Musatov et al. 2007;Shi and Clegg 2009). Increased food intake and body mass have been demonstrated in ovariectomized rats, with restoration of normal Fig. 6. Effect of Mcs3 on mammary gland development of females raised in the absence of males. Mammary carcinoma susceptible WF/NHsd, but not resistant WF.COP-Mcs3 J females had enhanced lobuloalveolar mammary gland histology at 12 weeks of age when raised in the absence of males. a-c) Representative H&E staining of WF/NHsd (left) and WF.COP-Mcs3 J (right) mammary glands at 4, 8, and 12 weeks of age. Images were saved at 40Â magnification using an Aperio ImageScope CS2. a) Mammary gland histology at 4 weeks is mixed TEBs and ductal structures embedded throughout an adipocyte matrix in both strains. b) Histology at 8 weeks is mixed TEB structures and maturing ductal structures with more pronounced stroma than at 4 weeks of age in both strains. c) Histological analysis at 12 weeks of age reveals Mcs3 J females have mature ducts and minimal lobuloalveolar structures (black arrows), which are representative of an adult virgin mammary gland. On the other hand, age-matched WF females display greatly enhanced lobuloalveolar structures. d) Quantitative analysis of % lobuloalveolar morphology in mammary glands at 12 weeks of age revealed increased lobuloalveolar area in susceptible WF compared to Mcs3 J females (n ¼ 3; 2-tailed unpaired t-test P < 0.0001).   Clegg et al. 2006). It remains to be determined if the Mcs3-mediated body mass difference, in the absence of males, is due to the disruption of endocrine signaling. An effect of Mcs3 on female body mass, dependent on the absence of males, is a classic example of a genotype-environment interaction (G Â E, Fletcher and Dudbridge 2014). Straindependent differences among grouped females have been reported (Bruce 1963). Our results could translate to human GWAS designs by indicating that if allelic effect sizes are masked by environmental effects (Dempfle et al. 2008). For example, a significant association of rs10483028 on chromosome 21q22.12 and breast cancer risk was detected in women with a body mass index below 25 kg/m 2 , and no association with disease was detected in women with a BMI of 30 kg/m 2 or higher (Schoeps et al. 2014). There is also suggestive evidence of a G Â E between the Mcs3orthologous human 15q25.1 region and smoking behavior for lung cancer risk. Numerous lung cancer risk risk-associated variants in this region also associate with smoking behavior and intensity. It stands to reason that the disease risk alleles marked by these variants were discovered because of genotypes influencing smoking behavior (VanderWeele et al. 2012; Yu et al. 2012). Thus, future studies of breast cancer risk warrant ultrafine mapping of rat Mcs3 and human 15q25.1-25.2 loci to identify causal disease variants and genes. These studies may need to consider a potential for G Â E interactions in study designs.
There is a cluster of olfactory receptor genes at the distal end of rat Mcs3. It is plausible that 1 or more of these genes could be involved in Mcs3 associated phenotype differences because it has been shown that effects of mammalian pheromones are prevented by excision of female olfactory bulbs (Parkes and Bruce 1961;Liberles 2014). Pheromonally induced changes in hormone signaling are known to modulate reproductive phenotypes. These include the induction of estrus cycling in noncycling females due to the presence of a male, known as the Whitten effect; pregnancy blocking of newly mated females due to a presence of strange or alien males, known as the Bruce effect; and suppression or inhibition of estrus cycling in grouped females, known as the Lee-Boot effect (Van Der Lee and Boot 1955;Bruce 1959;Whitten 1959;Ryan and Schwartz 1977). The latter was determined to cause entrance into pseudopregnancy and not short periods of anestrus. In our study, the advanced lobuloalveolar mammary gland morphology of susceptible females was . c) Orthologous syntenic region 2 contains part of 15q25.2 (chr15:83134545-84130720). Black horizontal bars represent the genomic track with intervals indicating genomic position beneath. Each lollipop color represents a different trait as indicated in the legend inset. Variants with multiple trait associations share a stem. consistent with pseudopregnancy morphologies and environmental conditions known to result in pseudopregnancies (Ryan and Schwartz 1977;Hvid et al. 2012). Pseudopregnancy is classically associated with failed fertilization, wherein the mechanical stimulus of copulation without fertilization results in a 10-to 12-day pseudopregnancy period characterized by a progestational state (Adler et al. 1970;Terkel 1986). However, during each estrus cycle, there is a brief period of sensitivity where ovarian corpus lutea can be activated and maintained (Terkel 1986). Socioenvironmental grouping of females after weaning without males is an initial corpus lutea-activating stimulus that allows a self-sustaining pseudopregnancy via elevated progesterone and positive feedback on luteotropic prolactin secretion (De Greef and Zeilmaker 1979;Terkel 1986).
In conclusion, rat Mcs3 was delimited to a 7.2-Mb locus that controls mammary carcinoma susceptibility, body mass, and mammary gland morphology. Concordance between orthologous human and rat loci for these traits provides strong evidence that human genetic and rat mechanistic studies are warranted. Additional studies, including further mapping of Mcs3 and functional characterization of positional candidates, will be needed to determine if Mcs3 pleiotropy is explained by a single locus or a cluster of independent subloci. The Mcs3 rat model developed in this study will be a valuable resource to identify causal genes and mechanisms of mammary cancer resistant that might be applicable to female breast cancer prevention. It will also be interesting to characterize the genotype by socioenvironmental interactions identified in this study, as findings may be applicable to human conditions including obesity, metabolic disorders, and mammary gland development windows of breast cancer susceptibility. Most importantly, these results provide strong rationale for further genetic analysis of the human orthologous locus for causal breast cancer susceptibility genes and variants.

Data availability
All data are presented within this article and supplementary files.
Supplemental material is available at G3 online.